P. gingivalis is a black-pigmented anaerobe that is associated with periodontal destruction in humans (Slots, 1977, Scand. J. Dent. Res. 85:247-354; Slots, 1986, J. Clin. Periodontal. 13:912-917; Socransky et al., 1990, p. 79-90 in J. D. Bader (ed.), Risk assessment in dentistry, University of North Carolina Dental Ecology, Chapel Hill). P. gingivalis is associated with several periodontal diseases including adult periodontitis, generalized juvenile periodontitis, periodontal abscesses, and refractory periodontitis. The tissues affected by periodontitis may include the gingival tissue, periodontal membrane, cementum, and the alveolar bone.
The organism is frequently found in low numbers when normal plaque is examined, or after treatment to reduce bacterial infection. Since it is unclear whether these low numbers represent a pathogenic threat, it is important to be able to quantitate, with accuracy and sensitivity, P. gingivalis in a given oral site in order to evaluate periodontal destruction potential. Current methods of detection of these fastidious organisms is dependent on culturing the bacteria or identifying the bacteria by immunofluorescence microscopy. P. gingivalis is difficult to cultivate on initial isolation due to its strict nutritional requirements and oxygen sensitivity (Zambon et all, 1988, J. Periodontol. 59:23-31). An example of current culture protocols involves inoculation onto enriched tryptic soy agar (ETSA) supplemented with 40 .mu.g/ml kanamycin, and incubation for seven days at 37.degree. C. in an anaerobic chamber containing 85% N.sub.2, 10% H.sub.2, and 5% CO.sub.2. Resultant isolates can then be identifed according to established procedures including gram-stain characteristics, cellular and colonial morphology, requirement for anaerobiosis, fermentation of specific sugars, and biochemical tests. Thus, the time required for growth and to complete identification can range from 7 to 21 days. Detection of P. gingivalis can also be accomplished by using immunofluorescence microscopy (Zambon et al., 1985, J. Periodontol. (Suppl.) 56:32-40 ). Although indirect immunofluorescence microscopy of P. gingivalis appears highly sensitive and specific for this organism, false positive reactions likely are due to the difficulties inherent in culturing this strictly anaerobic microorganism. These techniques, culturing the organism or detecting the organism by immunofluorescence, are both labor and time intensive.
Recent advances in molecular biology have provided several means for enzymatically amplifying nucleic acid sequences. Currently the most commonly used method, PCR (polymerase chain reaction, Cetus Corporation) involves the use of Taq Polymerase, known sequences as primers, and heating cycles which separate the replicating deoxyribonucleic acid (DNA) strands and exponentially amplify a gene of interest. Other amplification methods currently under development include LCR (ligase chain reaction, BioTechnica International) which utilizes DNA ligase, and a probe consisting of two halves of a DNA segment that is complementary to the sequence of the DNA to be amplified; enzyme QB replicase (Gene-Trak Systems) and a ribonucleic acid (RNA) sequence template attached to a probe complementary to the DNA to be copied which is used to make a DNA template for exponential production of complementary RNA; and NASBA (nucleic acid sequence-based amplification, Cangene Corporation) which can be performed on RNA or DNA as the nucleic acid sequence to be amplified.
Nucleic acid probes that are capable of hybridization with specific gene sequences have been used successfuly to detect specific pathogens in biological specimens at levels of sensitivity approaching 10.sup.3 -10.sup.4 organisms per specimen. Whole chromosome probes, developed from genomic DNA isolated from P. gingivalis and P. intermedius strains, have been used successfully in identifying P. gingivalis and P. intermedius in 74-77% of adult periodontitis samples compared to 21-26% identification by cultural analysis (Groves, 1990, pp. 236-237, in Gene Probe for Bacteria, eds. Macario and deMacario, Academic Press) However, the use of whole chromosome probes requires 10.sup.3 P. gingivalis cells for positive detection, and may require isolation of the strains from the clinical sample before use of the whole chromosome probes because of overlapping sequence homology and differences in the quantitative makeup of the oral flora by Porphyromonas species.
Cloned random gene fragments from closely related Bacteroides species have been used to detect and enumerate B. vulgaris in human feces (Kuritza et al., 1986, Appl. Environ. Microbiol. 51:385-390). Also, random fragments of chromosomal DNA from several Bacteroides species were cloned into plasmids from which RNA probes were developed (Groves et al., 1987, Diag. Microbiol. Infect. Dis. 7:273-278). The resultant RNA probes were used to detect isolated strains of Bacteroides species at a sensitivity of about 10.sup.6 cells. However, in systems using cloned random fragments, the specificity may be reduced because of shared sequences among Bacteroides species, as compared to amplification and hybridization of species-specific gene sequences.
Coupled with a method that allows for amplification of specific target DNA sequences, species-specific nucleic acid probes can greatly increase the level of sensitivity in detecting organisms in a clinical specimen. Use of these probes may allow direct detection without relying on prior culture and/or conventional biochemical identification techniques. The present invention is directed to primers which amplify species-specific sequences of known genes of P. gingivalis, and to probes which specifically hybridize with these amplified DNA fragments. By using the nucleic acid sequences of the present invention and according to the methods of the present invention, as few as one P. gingivalis organism may be detected in the presence of 10 .mu.g/ml extraneous DNA.